Chemical attachment of the hydrophilic polymer poly(ethylene glycol)(PEG), also known as-poly(ethylene oxide)(PEO), to molecules and surfaces is of great utility in biotechnology. In its most common form, PEG is a linear polymer terminated at each end with hydroxyl groups:HO—CH2CH2O—(CH2CH2O)n—CH2CH2—OHThis polymer can be represented in brief form as HO-PEG-OH where it is understood that the -PEG- symbol represents the following structural unit:—CH2CH2O—(CH2CH2O)n—CH2CH2—In typical form, n ranges from approximately 10 to approximately 2000.
PEG is commonly used as methoxy-PEG-OH, or mPEG in brief, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group that is subject to ready chemical modification.CH3O—(CH2CH2O)n—CH2CH2—OH mPEG
PEG is also commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, pentaerythritol and sorbitol. For example, the four-arm, branched PEG prepared from pentaerythritol is shown below;C(CH2—OH)4+n C2H4O—C[CH2O—(CH2CH2O)n—CH2CH2—OH]4
The branched PEGs can be represented in general form as R(-PEG-OH)n in which R represents the central “core” molecule, such as glycerol or pentaerythritol, and n represents the number of arms.
PEG is a much used polymer having the properties of solubility in water and in many organic solvents, lack of toxicity, and lack of immunogenicity. One use of PEG is to covalently attach the polymer to insoluble molecules to make the resulting PEG-molecule “conjugate” soluble. For example, Greenwald, Pendri and Bolikal in J. Org. Chem., 60, 331–336 (1995) have shown that the water-insoluble drug taxol, when coupled to PEG, becomes water soluble.
Davis et al. U.S. Pat. No. 4,179,337 describes proteins coupled to PEG and having enhanced blood circulation lifetime because of reduced rate of kidney clearance and reduced immunogenicity. The lack of toxicity of the polymer and its rapid clearance from the body are advantageous features for pharmaceutical applications. These applications and many leading references are described in the book by Harris (J. M. Harris, Ed., “Biomedical and Biotechnical Applications of Polyethylene Glycol Chemistry,” Plenum, New York, 1992).
To couple PEG to a molecule such as a protein it is necessary to use an “activated derivative” of the PEG having a functional group at the terminus suitable for reacting with some group on the surface or on the protein (such as an amino group). Among the many useful activated derivatives of PEG is the succinimidyl “active ester” of carboxymethylated PEG as disclosed by K. Iwasaki and Y. Iwashita in U.S. Pat. No. 4,670,417. This chemistry is illustrated with the active ester reacting with amino groups of a protein (the succinimidyl group is represented as NHS and the protein is represented as PRO-NH2):PEG-O—CH2—CO2—NHS+PRO-NH2—PEG-O—CH2—CO2—NH-PRO
Succinimidyl “active esters”, such as PEG-O-CH2—CO2—NHS, are commonly used forms of activated carboxylic acids, and they are prepared by reacting carboxylic acids with N-hydroxylsuccinimide.
Problems have arisen in the art. Some of the functional groups that have been used to activate PEG can result in toxic or otherwise undesirable residues when used for in vivo drug delivery. Some of the linkages that have been devised to attach functional groups to PEG can result in an undesirable immune response. Some of the functional groups do not have sufficient or otherwise appropriate selectivity for reacting with particular groups on proteins and can tend to deactivate the proteins.
PEG hydrogels, which are water-swollen gels, have been used for wound covering and drug delivery. PEG hydrogels are prepared by incorporating the soluble, hydrophilic polymer into a chemically crosslinked network or matrix so that addition of water produces an insoluble, swollen gel. Substances useful as drugs typically are not covalently attached to the PEG hydrogel for in vivo delivery. Instead, the substances are trapped within the crosslinked matrix and pass through the interstices in the matrix. The insoluble matrix can remain in the body indefinitely and control of the release of the drug can be somewhat imprecise.
One approach to preparation of these hydrogels is described in Embrey and Graham's U.S. Pat. No. 4,894,238, in which the ends of the linear polymer are connected by various strong, non-degradable chemical linkages. For example, linear PEG can be incorporated into a crosslinked network by reacting with a triol and a diisocyanate to form hydrolytically-stable (“non-degradable”) urethane linkages.
A related approach for preparation of non-degradable PEG hydrogels has been demonstrated by Gayet and Fortier in J. Controlled Release, 38, 177–184 (1996) in which linear PEG was activated as the p-nitrophenylcarbonate and crosslinked by reaction with a protein, bovine serum albumin. The linkages formed are hydrolytically-stable urethane groups.
N. S. Chu, in U.S. Pat. No. 3,963,805, describes non-degradable PEG networks have been prepared by random entanglement of PEG chains with other polymers formed by use of free radical initiators mixed with multifunctional monomers. P. A. King, in U.S. Pat. No. 3,149,006, describes the preparation of non-degradable PEG hydrogels by radiation-induced crosslinking of high molecular weight PEG.
Nagaoka et al., in U.S. Pat. No. 4,424,311, describe PEG hydrogels prepared by copolymerization of PEG methacrylate with other comonomers such as methyl methacrylate. This vinyl polymerization will produce a polyethylene backbone with PEG attached. The methyl methacrylate comonomer is added to give the gel additional physical strength.
Sawhney, Pathak and Hubbell in Macromolecules, 26, 581 (1993) describe the preparation of block copolymers of polyglycolide or polylactide and PEG that are terminated with acrylate groups, as shown below:CH2═CH—CO—(O—CH2—CO)n-PEG-(O—CH2—CO)n—O—CO—CH═CH2In the above formula, the glycolide blocks are the —O—CH2—CO— units; addition of a methyl group to the methylene gives a lactide block; n can be multiples of 2. Vinyl polymerization of the acrylate groups produces an insoluble, crosslinked gel with a polyethylene backbone. The polylactide or polyglycolide segments of the polymer backbone, being ester groups, are susceptible to slow hydrolytic breakdown, with the result that the crosslinked gel undergoes slow degradation and dissolution.
Substantial non-PEG elements are introduced into the hydrogel. Non-PEG elements tend to introduce complexity into the hydrogel and degradation and dissolution of the matrix can result in undesirable or toxic components being released into the blood stream when the hydrogels are used in vivo for drug delivery.
It would be desirable to provide alternative PEG hydrogels that are suitable for drug delivery and that have unique properties that could enhance drug delivery systems.